Crop Biotechnology - American Chemical Society

Chapter 12

Reduction of Aflatoxin Contamination in Peanut: A Genetic Engineering Approach 1

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P. Ozias-Akins , H. Yang , R. Gill , H. Fan , and R. E. Lynch

Downloaded by UNIV OF ARIZONA on July 22, 2015 | http://pubs.acs.org Publication Date: August 7, 2002 | doi: 10.1021/bk-2002-0829.ch012

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Department of Horticulture, The University of Georgia Tifton Campus, Tifton, GA 31793-0748 C r o p Protection and Management Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Coastal Plain Experiment Station, Tifton, GA 31793 2

Development of methods for the introduction of foreign genes into peanut provides an adjunct means to conventional breeding for genetic improvement of the crop for disease resistance. Transformation of peanut is based on microprojectile bombardment of repetitive embryogenic tissue cultures. These cultures can be initiated most efficiently from immature cotyledons or mature embryo axes by culture of the explants on auxin (picloram)-supplemented media. Somatic embryos developing from the primary cultures will undergo repetitive growth when maintained on picloram. Removing auxinfromthe medium and adding a cytokinin will promote the development of shoots from the somatic embryos. Transformation is accomplished by bombardment of embryogenic cultures with DNA-coated gold particles and selection of transgenic lines on the antibiotic hygromycin. Fewer than 5% of the plants recovered from hygromycin-resistantlines are escapesfromselection. Although

© 2002 American Chemical Society

In Crop Biotechnology; Rajasekaran, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2002.

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transformation of peanut by this method is slow, taking approximately 12-14 months, it is highly reproducible and genotypeindependent. Aflatoxin contamination of peanut seeds originates with the contaminating fungus, Aspergillus flavus, which is an opportunistic saprophyte. Using genetic engineering, we have initiated a three-tiered approach to reduce 1) access of the fungus to the peanut pod, 2) fungal growth, and 3) aflatoxin biosynthesis. This approach encompasses the introduction of insect and fungal resistance genes, as well as genes whose products may interfere with aflatoxin production.

Peanut (Arachis hypogaea L.) is an important source of oil and is widely used in the confectionary industry, in candies and in peanut butter. However, unlike soybean (Glycine max) which in the US is grown on over 70 million acres, peanut is cultivated on only 1.5 million acres (1). Due to its status as a minor crop in the US, peanut has not received input comparable to soybean for the development of improved cultivars through the application of biotechnology. Although there are many potential targets for the improvement of peanut by genetic engineering, most notably disease resistance, pest control, and oil composition, one of the most serious industry-wide problems is aflatoxin contamination of peanut seeds which are used in food and feed products. Aflatoxin is a my cotoxin that is produced by Aspergillusflavus and A. parasiticus, two fungal species that are prevalent in soils. Because peanut pods develop underground, Aspergillus can easily invade visibly damaged pods (damaged by insects or mechanical means), but it also may be found infrequently in apparently undamaged pods. Production of aflatoxin, a secondary metabolite in the saprophytic fungus, is enhanced during plant stress induced by drought and high soil temperatures. Aflatoxin has been identified as a carcinogen; therefore, levels in food products are restricted by the Food and Drug Administration to 20 ppb except for milk which has an action level of 0.5 ppb. These regulations require that aflatoxigenic fimgi and aflatoxin levels be monitoredfromthe buying point through peanut processing. Approaches to control preharvest aflatoxin contamination range from biocontrol with atoxigenic strains, to modified cultural practices and the introduction of genetic resistance (2). Identification of quantitative genetic resistance within the gene pool holds promise, although screening methods require extensive replication to produce meaningful results (5). In addition to accessing potential resistance from the A. hypogaea gene pool, genetic engineering to introduce genes for fungal resistance, insect resistance (since aflatoxin levels are correlated with insect damage), or reduction of aflatoxin biosynthesis may be a


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realistic adjunct to traditional breeding. Our goal has been to develop a reproducible, genotype-independent transformation system for peanut that can be used to test the efficacy of foreign genes for reducing aflatoxin contamination.


A Reproducible, Genotype-Independent Transformation System for Peanut Most transformation systems, whether mediated by biological means (Agrobacterium tumefaciens) or by physical methods of free DNA uptake (microprojectile bombardment, electroporation, silica carbide whiskers, etc.), rely on efficient plant regenerationfromtissue cultures of the species of interest (4). In peanut, dedifferentiation oftissues into a true callus phase (unorganized growth and cell division) eliminates regeneration ability. A variety of immature tissues can, however, be induced to directly form shoot primordia or somatic embryos when plated on the appropriate growth regulators. In our hands, induction of somatic embryos from immature cotyledons or embryo axes of peanut seeds can be accomplished with all genotypes tested when the explant tissues are plated on a standard tissue culture medium (5) that has been supplemented with the growth regulator, picloram (6-8; Fig. 1). Somatic embryos form directlyfromthe explanted tissue without an intervening callus phase. The embryogenic growth typically originatesfromthe portion of the cotyledon proximal to the cotyledonary node or from the epicotyledonary portion of the embryo axis either between the first true leaves and the axillary buds at the cotyledonary node or from the young leaf primordia. The embryogenic tissues are capable of repetitive embryogenesis when subcultured at regular intervals onto medium of the same composition. Whole peanut plants can be regeneratedfrommature somatic embryos using a 2- to 3-step protocol (9). When root and shoot poles of a somatic embryo do not develop and elongate simultaneously, the shoot can be excised and easily rooted on medium containing 0.2 mg/1 naphthaleneacetic acid. Although no tap root is subsequently present to support growth of the plant, numerous adventitious roots allow a rapid transitionfromagar-based culture medium to potting mix. A uniform, readily regenerable tissue culture facilitates plant transformation. The embryogenic tissue culture should consist primarily of translucent, smoothsurfaced somatic embryos between globular and early cotyledonary stages of development in order to increase the probability of recovering transgenic cell lines. Foreign genes can be introduced into uniform, embryogenic cultures of peanut by microprojectile bombardment(9). We typically bombard cultures (10-14 days after subculture) with DNA-coated gold particles accelerated by bursting of an 1800 psi rupture disc with helium pressure. Just prior to bombardment, tissue pieces are arranged in a 2 cm-diameter circle in the center of the culture dish where they



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Figure 1. Immature embryos (A) cm be excisedfromthe surrounding seedcoat tissue and separated into cotyledons and embryo axis. Cultured cotyledons will form somatic embryos at their base (B) which can be subcultured to produce repetitive embryogenic cultures (C). Removing picloramfromthe culture medium will allow somatic embryos to develop further (D). Organogenic cultures in which shoots, rather than somatic embryos, develop can be induced on media containing virions cytokinins (E). (B and D are reproduced with permissionfromreference 8. Copyright 1992 Urban and Fischer Verlag)



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remain until 2-3 days post-bombardment. If the tissues were bombarded with a construct containing the reporter gene, P-glucuronidase (GUS), pieces of tissue can be stained to assess the effectiveness of the bombardment conditions (Fig. 2). Tissues cultured under non-selective conditions and stained for GUS activity after one month may show the development of a transgenic cell lineage (Fig. 2). Since the GUS assay is destructive, such lineages cannot be selectively transferred, although the use of a non-destructive reporter such as the greenfluorescentprotein might allow small sectors to be manually recovered (10). We have opted for antibiotic selection of stably transformed cells which is most effective with hygromycin, The hygromycin phosphotransferase gene (hph) is expressed sufficiently in embryogenic tissues when driven by either the CaMV35S promoter or the potato ubiquitin 3 promoter (/ /). Hygromycin resistance is clearly expressed not only during selection for embryogenic tissues//! vitro, but also during rooting and in young, mature leaves (Fig. 3). Regeneration of plantsfromtransgenic cell lines is possible if the cell lines have remained clearly embryogenic. Morphologically normal,floweringplants usually are obtainedfromtransgenic somatic embryos. However, peanut appears to be unusually susceptible to the deleterious effects of the tissue culture and transformation process with regard to flowering and fertility. Although plant regeneration occurs readilyfromembryogenic cultures even after extended culture duration, many of the primary regenerants display delayedfloweringand partial or complete sterility. For this reason, it is recommended that tissues be maintained only as long as necessary to establish a sufficient number of uniform cultures to carry out bombardments within 4-9 months of culture initiation. Fourteen months typically are required to complete the transformation cycle from seed to seed including culture initiation, bombardment and selection, regeneration, acclimitization and maturation. Transformation of peanut using the general process outlined above has been successfully repeated in our lab as well as others (9, 12-16).

Application of Peanut Genetic Engineeringtothe Problem of Aflatoxin Contamination We are taking a three-tiered approach to reduction of pre-harvest aflatoxin contamination, using genetic engineering, that addresses insect damage, fungal growth and aflatoxin biosynthesis. Only our efforts to control insect damage in peanut have progressed to the point of efficacy testing in the field, thus they will be described in the most detail below. It has been clearly documented that aflatoxin contamination is positively correlated with insect damage (17). In peanut, the insect pest most commonly associated with aflatoxin contamination is the lesser cornstalk borer (Elasmopalpus lignosellus; LCB). The larvae of LCB often tunnel into



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Figure 2. Bombardment with the reporter gene ^-glucuronidase allows an assessment oftransient expression (A) orformation ofstably transformed sectors (B).

Figure J. The antibiotic, hygromycin can be used to select transgenic embryogenic tissues in liquid (A) or agar (B) medium, at rooting (C), or during screening ofleaf discsfromregeneratedplants andprogeny (D). (B reproduced with permissionfromreference 9. Copyright 1993 ElsevierScience) In Crop Biotechnology; Rajasekaran, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2002.


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peanut stems and feed on pods developing underground (18). Damage to pods can be due to penetration of the pod wall or extensive scarification of the pod surface (Fig. 4). LCB thrives in hot, dry weather when soil temperatures are high, and these also are the most favorable conditions for aflatoxin production. To control the amount of pod damage attributed to LCB, the most effective means may be by host plant resistance since soil insecticides have a variable period of residual activity. LCB is a lepidopteran insect pest and thus susceptible to several of the insecticidal crystalline proteins found in the soil bacterium, Bacillus thuringiensis (19). Peanut genotypes transformed with Bt cryIA(c) are resistant to foliar damage by LCB when the larvae are forced to feed on leaves in vitro (12; Fig. 4). Field resistance to insect damage to foliage and pods has been observed (Fig. 4). Four transgenic lines recovered by transformation of cultivar Marcl in 1995 have been carried forward duringfieldtesting. These lines initially were selected based on in vitro assays at the TI generation (Table 1). One of the lines does not carry an intact Bt cryIA(c) gene as shown by PCR analysis of multiple T3 individuals (Fig. 5). CryIA(c)expressing and control lines are beingfieldtested in 2000 for both LCB resistance as well as aflatoxin reduction.

Table I. L C B Bioassaly on Transgenic Peanut Line

Survival (%)

Weight (mg)

Damage (%)

22 (89-5-23)

0

0

Crop Biotechnology - American Chemical Society

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